MicroRNA-31 is required for astrocyte specification

Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis

O-GlcNAc Modification of NFκB p65 Inhibits TNF-α-Induced Inflammatory Mediator Expression in Rat Aortic Smooth Muscle Cells

BACKGROUND: We have shown that glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatment augments O-linked-N-acetylglucosamine (O-GlcNAc) protein modification and attenuates inflammatory mediator expression, leukocyte infiltration and neointima formation in balloon injured rat carotid arteries and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response. NFκB signaling has been shown to mediate the expression of inflammatory genes and neointima formation in injured arteries. Phosphorylation of the p65 subunit of NFκB is required for the transcriptional activation of NFκB. This study tested the hypothesis that GlcN or PUGNAc treatment protects vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress by enhancing O-GlcNAcylation and inhibiting TNF-α induced phosphorylation of NFκB p65, thus inhibiting NFκB signaling. METHODOLOGY/PRINCIPAL FINDINGS: Quiescent rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (10(-4) M) or vehicle and then stimulated with TNF-α (10 ng/ml). Both treatments inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2β and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1 and P-Selectin]. Both treatments inhibited TNF-α induced NFκB p65 activation and promoter activity, increased NFκB p65 O-GlcNAcylation and inhibited NFκB p65 phosphorylation at Serine 536, thus promoting IκBα binding to NFκB p65. CONCLUSIONS: There is a reciprocal relationship between O-GlcNAcylation and phosphorylation of NFκB p65, such that increased NFκB p65 O-GlcNAc modification inhibits TNF-α-Induced expression of inflammatory mediators through inhibition of NFκB p65 signaling. These findings provide a mechanistic basis for our previous observations that GlcN and PUGNAc treatments inhibit inflammation and remodeling induced by acute endoluminal arterial injury

Protective Role of STAT3 in NMDA and Glutamate-Induced Neuronal Death: Negative Regulatory Effect of SOCS3

<div><p>The present study investigates the involvement of the IL-6 family of cytokines, activation of the transcription factor Signal Transducer and Activator of Transcription-3 (STAT3), and the role of Suppressor Of Cytokine Signaling-3 (SOCS3) in regulating excitotoxic neuronal death <em>in vitro</em>. Biochemical evidence demonstrates that in primary cortical neurons and SH-SY5Y neuroblastoma cells, IL-6 cytokine family members, OSM and IL-6 plus the soluble IL-6R (IL-6/R), prevent NMDA and glutamate-induced neuronal toxicity. As well, OSM and IL-6/R induce tyrosine and serine phosphorylation of STAT3 in primary cortical neurons and SH-SY5Y cells. Studies using Pyridine 6 (P6), a pan-JAK inhibitor, demonstrate that the protective effect of OSM and IL-6/R on neuronal death is mediated by the JAK/STAT3 signaling pathway. In parallel to STAT3 phosphorylation, OSM and IL-6/R induce SOCS3 expression at the mRNA and protein level. P6 treatment inhibits SOCS3 expression, indicating that STAT3 is required for OSM and IL-6/R-induced SOCS3 expression. Lentiviral delivery of SOCS3, an inhibitor of STAT3 signaling, into primary neurons and SH-SY5Y cells inhibits OSM and IL-6/R-induced phosphorylation of STAT3, and also reverses the protective effect of OSM and IL-6/R on NMDA and glutamate-induced neurotoxicity in primary cortical neurons. In addition, treatment with IL-6 cytokines increases expression of the anti-apoptotic protein Bcl-xL and induces activation of the Akt signaling pathway, which are also negatively regulated by SOCS3 expression. Thus, IL-6/R and OSM-induced SOCS3 expression may be an important factor limiting the neuroprotective effects of activated STAT3 against NMDA and glutamate-induced neurotoxicity.</p> </div

P6 Reverses the Protective Effect of IL-6 Cytokines on NMDA-induced Toxicity.

<p><b>A</b>–<b>B</b>, Primary cortical neurons (A) or SH-SY5Y cells (B) were pretreated with P6 (0.5 µM) for 1 h, and then grown in the presence of P6 and IL-6 plus sIL-6R or OSM for 1 h, and then in the presence of cytokine, P6, plus NMDA for an additional 24 h. Cell viability was determined by the MTT reduction assay. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. ***p<0.001 compared to control; **p<0.001, *p<0.01, and ♦p<0.05 compared to NMDA; and ?p<0.001 and #p<0.01 compared to NMDA plus cytokines. <b>C</b>–<b>D</b>, P6 Inhibits IL-6 Cytokine-induced STAT3 Activation. Primary neurons (C) or SH-SY5Y cells (D) were grown with P6 for 1 h and where indicated, grown in the presence of OSM or IL-6 plus sIL-6R and P6. The levels of phosphorylated STAT3 Tyr705, phosphorylated STAT3 Ser727, and total STAT3 were analyzed at the indicated time points. The densitometric ratios of P-STAT3 Tyr705 or P-STAT3 Ser727 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in four separate experiments. *<i>p</i><0.001 and **p<0.01 compared to control; and ?p<0.001, #p<0.01, and &p<0.05 compared to IL-6 plus IL-6R or OSM alone.</p

Induction of SOCS3 Expression by IL-6 Cytokines in Primary Neurons and SH-SY5Y Cells.

<p><b>A,</b> Primary neurons were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for the times indicated, and total RNA was analyzed by qRT-PCR. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. **p<0.001 compared to control. <b>B</b>, SH-SY5Y cells were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for the times indicated, and cell lysates immunoblotted with SOCS3 and GAPDH antibodies. <b>C</b>–<b>E,</b> Inhibition of the JAK/STAT Pathway Inhibits SOCS3 Expression. <b>C, D</b>, Primary cortical neurons (C) or SH-SY5Y cells (D) were pretreated with P6 (0.5 µM) for 1 h, and then treated with IL-6 plus sIL-6R or OSM in the presence of continued P6 exposure, and total mRNA was analyzed by qRT-PCR. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. *p<0.001 compared to control; **p<0.001 compared to IL-6 plus IL-6R or OSM alone. <b>E</b>, SH-SY5Y cells were grown in the absence or presence of P6 pretreatment for 1 h and then grown in the presence of P6 and IL-6 plus sIL-6R or OSM and protein levels of SOCS3 were analyzed. The densitometric ratios of SOCS3 versus GAPDH were calculated. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to control; ?p<0.001 compared to IL-6 plus IL-6R or OSM alone.</p

Proposed Model for SOCS3 Contribution to Excitoxicity in Neurons. A

<p>, IL-6/R or OSM activates the JAK/STAT3 pathway, which increases Bcl-xL expression and causes activation of the Akt signaling pathway in a STAT3-dependent manner. Increased Bcl-xL expression and binding to Bax, as well as Akt activation inhibits NMDA or glutamate-induced neuronal death. <b>B</b>, IL-6/R and OSM also induce SOCS3 expression in a STAT3-dependent fashion. SOCS3, in turn, modulates IL-6/R or OSM induced Bcl-xL expression and Akt signaling pathway in a negative regulatory manner, which contributes, in part, to NMDA or glutamate-induced neuronal death.</p

SOCS3 Inhibits STAT3 Activation in Primary Neurons and SH-SY5Y Cells.

<p><b>A</b>–<b>B</b>, Primary neurons (A) or SH-SY5Y cells (B) were infected with Lenti-control (−) or Lenti-SOCS3 (+). After infection, IL-6 plus sIL-6R or OSM was added to primary cortical neurons or SH-SY5Y cells for up to 1 h, and protein lysates were subjected to immunoblot analysis with antibodies against phosphorylated STAT3 Tyr705, total STAT3, SOCS3 and GAPDH. The densitometric ratios of P-STAT3 Tyr705 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to Lenti-control infected cells left untreated; ?p<0.001, and ??p<0.01 compared to Lenti-control infected cells treated with IL-6 plus IL-6R or OSM. <b>C</b>, SH-SY5Y cells were infected with control or shSOCS3 lentivirus, treated with OSM for 1 h, and total RNA was analyzed by qRT-PCR for SOCS3 expression. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. *p<0.001 compared to control; **p<0.001 compared to OSM treatment of control lentivirus infected culture. <b>D</b>, After infection of SH-SY5Y cells with pGipz (control) or shSOCS3 lentivirus, IL-6 plus sIL-6R or OSM was added for the times indicated and cell lysates immunoblotted as described. The densitometric ratios of P-STAT3 Tyr705 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to control cultures infected with pGipz; ?p<0.001, and ??p<0.01 compared to IL-6 plus IL-6R or OSM treatment of pGipz lentivirus infected cultures.</p

Regulation of the Akt Signaling Pathway by IL-6 Cytokines and SOCS3.

<p><b>A</b>–<b>B</b>, Primary neurons (A) were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml), or SH-SY5Y cells (B) were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) in the absence or presence of P6 (0.5 µM) pretreatment for 1 h, and protein lysates subjected to immunoblot analyses with antibodies against P-Akt, total Akt and GAPDH. P6 was also present for indicated time points of cytokine exposure. The densitometric ratios of P-Akt versus total Akt were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001, **p<0.01, and ***p<0.05 compared to control; ?p<0.001, &p<0.01, and ??p<0.05 compared to IL-6 plus IL-6R or OSM alone. <b>C</b>, SH-SY5Y cells were pretreated for 2 h with the PI3-Kinase inhibitors LY294002 (10 µM) or Wortmannin (1 µM), followed by treatment with IL-6 plus sIL-6R or OSM for 30 min with continued LY294002 or Wortmannin exposure, and protein levels of phosphorylated STAT3 Tyr705 and total STAT3 were analyzed. The densitometric ratios of P-STAT3 Tyr705 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to control. <b>D</b>, SH-SY5Y cells were infected with Lenti-control (−) or Lenti-SOCS3 (+). After infection, IL-6 plus sIL-6R or OSM was added to SH-SY5Y cells for the times indicated, and protein lysates subjected to immunoblot analyses with antibodies against P-Akt, total Akt, SOCS3 and GAPDH. The densitometric ratios of P-Akt versus total Akt were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to Lenti-control lentivirus infected cultures left untreated; ?p<0.001 compared to IL-6 plus IL-6R or OSM treatment of control lentivirus infected cells.</p

RNAdigest: A Web-Based Tool for the Analysis and Prediction of Structure - Specific RNAse Digestion Results

<div><p>Despite recent developments in analyzing RNA secondary structures, relatively few RNA structures have been determined. To date, many investigators have relied on the traditional method of using structure-specific RNAse enzymes to probe RNA secondary structures. However, if these data were combined with novel computational approaches, investigators would have an informative and valuable tool for RNA structural analysis. To this end, we created the web server “RNAdigest.” RNAdigest uses mfold RNA structural models in order to predict the results of RNAse digestion experiments. Furthermore, RNAdigest also utilizes both RNA sequence and the experimental digestion patterns to formulate the constraints for predicting secondary structures of the RNA. Thus, RNAdigest allows for the structural interpretation of RNAse digestion experiments. Overall, RNAdigest simplifies RNAse digestion result analyses while allowing for the identification of unique fragments. These unique fragments can then be used for testing predicted mfold structures and for designing structural-specific DNA/RNA probes.</p></div